Body composition analysis has become a useful tool in both clinical and research settings. Its use in the pediatric population is complicated by the rapid periods of growth and physical development that are characteristic of infancy, childhood, and adolescence. A thorough understanding of the changing nature of body composition during this time is essential for choosing the most appropriate measurement technique for a given individual, population, or clinical question. Growing evidence suggests that tissues such as fat, muscle, and bone are intimately involved in the regulation of whole body energy metabolism. This knowledge, when coupled with advancements in imaging techniques such as MRI and PET-CT, offers the possibility of developing new models of “functional” body composition. These models may prove to be especially important when assessing malnutrition and metabolic risk in patients with chronic disease.
Body Composition; Obesity; Fat Mass Index; Lean Body Mass Index; Body Mass Index; Metabolic Syndrome
State-specific healthcare-associated infection (HAI) cost estimates have not been calculated to guide Department of Public Health efforts and investments.
We completed a cost identification study by conducting a survey of 117 acute care hospitals in NC hospitals to collect surveillance data on patient-days, device-days, and surgical procedures during a one-year. We then calculated expected rates and direct hospital costs of surgical site infections (SSI), C. difficile infection and 3 selected device-related HAIs for hospitals and the entire state using reference datasets such as the National Heatlhcare Safety Network (NHSN).
In total, 67 (53%) hospitals responded to the survey. The median bed size of respondent hospitals was 140 (IQR 66–350). A “standard” NC hospital diagnoses approximately 100 HAI each year with estimated costs of $985,000 to $2.7 million. The most common HAI was SSI (73%). Costs related to SSI accounted for 87% to 91% of overall costs. In total, the overall direct annual cost of these five selected HAIs was estimated to be between $124.1 and $347.8 million in 2009 for the State of NC.
Using conservative estimates, HAI led to costs of more than $100 million in acute care hospitals in the State of NC in 2009. The majority of costs were due to SSI.
Brugada syndrome is a rare cardiac arrhythmia disorder, causally related to SCN5A mutations in around 20% of cases1–3. Through a genome-wide association study of 312 individuals with Brugada syndrome and 1,115 controls, we detected 2 significant association signals at the SCN10A locus (rs10428132) and near the HEY2 gene (rs9388451). Independent replication confirmed both signals (meta-analyses: rs10428132, P = 1.0 × 10−68; rs9388451, P = 5.1 × 10−17) and identified one additional signal in SCN5A (at 3p21; rs11708996, P = 1.0 × 10−14). The cumulative effect of the three loci on disease susceptibility was unexpectedly large (Ptrend = 6.1 × 10−81). The association signals at SCN5A-SCN10A demonstrate that genetic polymorphisms modulating cardiac conduction4–7 can also influence susceptibility to cardiac arrhythmia. The implication of association with HEY2, supported by new evidence that Hey2 regulates cardiac electrical activity, shows that Brugada syndrome may originate from altered transcriptional programming during cardiac development8. Altogether, our findings indicate that common genetic variation can have a strong impact on the predisposition to rare diseases.
We estimated the incidence of watery diarrhea in the community before and after introduction of the pentavalent rotavirus vaccine in León, Nicaragua. A random sample of households was selected before and after rotavirus vaccine introduction. All children < 5 years of age in selected households were eligible for inclusion. Children were followed every 2 weeks for watery diarrhea episodes. The incidence rate was estimated as numbers of episodes per 100 child-years of exposure time. A mixed effects Poisson regression model was fit to compare incidence rates in the pre-vaccine and vaccine periods. The pre-vaccine cohort (N = 726) experienced 36 episodes per 100 child-years, and the vaccine cohort (N = 826) experienced 25 episodes per 100 child-years. The adjusted incidence rate ratio was 0.60 (95% confidence interval [CI] 0.40, 0.91) during the vaccine period versus the pre-vaccine period, indicating a lower incidence of watery diarrhea in the community during the vaccine period.
Although the influenza vaccine is recommended for end-stage renal disease (ESRD) patients, little is known about its effectiveness. Observational studies of vaccine effectiveness (VE) are challenging because vaccinated persons may be healthier than unvaccinated persons.
Using United States Renal Data System data, we estimated VE for influenza-like illness (ILI), influenza/pneumonia hospitalization, and mortality in adult, hemodialysis patients using a natural experiment created by year-to-year variation in the match of the influenza vaccine to the circulating virus. Matched (1998, 1999, 2001) and unmatched (1997) years among vaccinated patients were compared using Cox proportional hazards models. Ratios of hazard ratios compared vaccinated patients between two years and unvaccinated patients between two years. VE was calculated as 1 - effect measure.
Vaccination rates were <50% each year. Conventional analysis comparing vaccinated to unvaccinated patients produced average VE estimates of 13%, 16%, and 30% for ILI, influenza/pneumonia hospitalization and mortality respectively. When restricted to the pre-influenza period, results were even stronger, indicating bias. The pooled ratio of HRs comparing matched seasons to a placebo season resulted in a VE of 0% (95% CI: −3,2%) for ILI, 2% (95% CI: −2,5%) for hospitalization, and 0% (95% CI: −3,3%) for death.
Relative to a mismatched year, we found little evidence of increased VE in subsequent, well-matched years, suggesting that the current influenza vaccine strategy may have a smaller effect on morbidity and mortality in the ESRD population than previously thought. Alternate strategies (high dose vaccine, adjuvanted vaccine, multiple doses) should be investigated.
Influenza vaccines; vaccine effectiveness; bias (epidemiology); renal dialysis; cohort studies
Staphylococcus aureus is a common cause of bacteremia, with a substantial impact on morbidity and mortality. Because of increasing rates of methicillin-resistant Staphylococcus aureus, vancomycin has become the standard empirical therapy. However, beta-lactam antibiotics remain the best treatment choice for methicillin-susceptible strains. Placing patients quickly on the optimal therapy is one goal of antimicrobial stewardship. This retrospective, observational, single-center study compared 33 control patients utilizing only traditional full-susceptibility methodology to 22 case patients utilizing rapid methodology with CHROMagar medium to detect and differentiate methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains hours before full susceptibilities were reported. The time to targeted therapy was statistically significantly different between control patients (mean, 56.5 ± 13.6 h) and case patients (44.3 ± 17.9 h) (P = 0.006). Intensive care unit status, time of day results emerged, and patient age did not make a difference in time to targeted therapy, either singly or in combination. Neither length of stay (P = 0.61) nor survival (P = 1.0) was statistically significantly different. Rapid testing yielded a significant result, with a difference of 12.2 h to targeted therapy. However, there is still room for improvement, as the difference in time to susceptibility test result between the full traditional methodology and CHROMagar was even larger (26.5 h). This study supports the hypothesis that rapid testing plays a role in antimicrobial stewardship by getting patients on targeted therapy faster.
To determine the effectiveness of an automated ultraviolet-C (UV-C) emitter against vancomycin-resistant enterococci (VRE), Clostridium difficile, and Acinetobacter spp. in patient rooms.
Prospective cohort study.
Two tertiary care hospitals.
Convenience sample of 39 patient rooms from which a patient infected or colonized with 1 of the 3 targeted pathogens had been discharged.
Environmental sites were cultured before and after use of an automated UV-C-emitting device in targeted rooms but before standard terminal room disinfection by environmental services.
In total, 142 samples were obtained from 27 rooms of patients who were colonized or infected with VRE, 77 samples were obtained from 10 rooms of patients with C. difficile infection, and 10 samples were obtained from 2 rooms of patients with infections due to Acinetobacter. Use of an automated UV-C-emitting device led to a significant decrease in the total number of colony-forming units (CFUs) of any type of organism (1.07 log10 reduction; P < .0001), CFUs of target pathogens (1.35 log10 reduction; P < .0001), VRE CFUs (1.68 log10 reduction; P < .0001), and C. difficile CFUs (1.16 log10 reduction; P < .0001). CFUs of Acinetobacter also decreased (1.71 log10 reduction), but the trend was not statistically significant (P = .25). CFUs were reduced at all 9 of the environmental sites tested. Reductions similarly occurred in direct and indirect line of sight.
Our data confirm that automated UV-C-emitting devices can decrease the bioburden of important pathogens in real-world settings such as hospital rooms.
Seventy-eight blood cultures with a Gram stain result of Gram-positive cocci in pairs and/or chains were evaluated with the Nanosphere Verigene Gram-positive blood culture (BC-GP) assay. The overall concordance of the assay with culture was 89.7% (70/78 cultures), allowing for the development of a targeted treatment algorithm.
To better understand the antibiotic prescribing process in assisted living (AL) communities given the growing rates of antibiotic resistance.
Four AL communities in North Carolina.
AL residents who received antibiotics (n=30) from October 20, 2010 to March 31, 2011, and their primary family member, staff, and the prescribing medical provider.
Semi-structured interviews conducted about prescribing included the information available at the time of prescribing and the perceptions of the quality of communication among providers, staff, residents and family members about the case. Providers were asked an open-ended question regarding how to improve the communication process related to antibiotic prescribing for AL residents.
Among 30 cases of antibiotic prescriptions, providers often had limited information about the case and lacked familiarity with the residents, the residents’ families, and/or staff. In addition, they felt cases were less severe and less likely to need an antibiotic than did residents, families, and staff. Providers identified several ways to improve the communication process including better written documentation and staff/family presence.
In our small sample of AL communities, providers faced an array of challenges in making antibiotic prescribing decisions. Our work confirms the complex nature of antibiotic prescribing in AL communities and reveals further work is needed to determine how to improve the appropriateness of antibiotic prescribing.
Assisted Living; antibiotic; older adults; prescribing
While the majority of healthcare in the US is provided in community hospitals, the epidemiology and treatment of bloodstream infections in this setting is unknown.
Methods and Findings
We undertook this multicenter, retrospective cohort study to 1) describe the epidemiology of bloodstream infections (BSI) in a network of community hospitals and 2) determine risk factors for inappropriate therapy for bloodstream infections in community hospitals. 1,470 patients were identified as having a BSI in 9 community hospitals in the southeastern US from 2003 through 2006. The majority of BSIs were community-onset, healthcare associated (n = 823, 56%); 432 (29%) patients had community-acquired BSI, and 215 (15%) had hospital-onset, healthcare-associated BSI. BSIs due to multidrug-resistant pathogens occurred in 340 patients (23%). Overall, the three most common pathogens were S. aureus (n = 428, 28%), E. coli (n = 359, 24%), coagulase-negative Staphylococci (n = 148, 10%), though type of infecting organism varied by location of acquisition (e.g., community-acquired). Inappropriate empiric antimicrobial therapy was given to 542 (38%) patients. Proportions of inappropriate therapy varied by hospital (median = 33%, range 21–71%). Multivariate logistic regression identified the following factors independently associated with failure to receive appropriate empiric antimicrobial therapy: hospital where the patient received care (p<0.001), assistance with ≥3 ADLs (p = 0.005), Charlson score (p = 0.05), community-onset, healthcare-associated infection (p = 0.01), and hospital-onset, healthcare-associated infection (p = 0.02). Important interaction was observed between Charlson score and location of acquisition.
Our large, multicenter study provides the most complete picture of BSIs in community hospitals in the US to date. The epidemiology of BSIs in community hospitals has changed: community-onset, healthcare-associated BSI is most common, S. aureus is the most common cause, and 1 of 3 patients with a BSI receives inappropriate empiric antimicrobial therapy. Our data suggest that appropriateness of empiric antimicrobial therapy is an important and needed performance metric for physicians and hospital stewardship programs in community hospitals.
Diarrhoea remains an important cause of mortality and morbidity among children in Nicaragua. As the majority of diarrhoeal cases are treated at home and appropriate household management can lessen severity of diarrhoea, the objective of this study was to examine household management of childhood diarrhoea. A simple random sample of households was selected from the Health and Demographic Surveillance Site-León. Parents or caretakers of children below five years of age, who developed diarrhoea (n=232), were surveyed about household diarrhoea management practices in 2011. Fifty-seven percent of children received oral rehydration therapy (ORT) in the home prior to visiting any health facility. We encountered certain practices in contradiction with WHO recommendations for the management of diarrhoea in communities: 41% of children were offered protein-rich foods less frequently during diarrhoeal episodes, 20% of children were nursed less frequently or not at all during diarrhoeal episodes, and zinc supplementation was recommended at only 39% of visits with healthcare providers. Our findings provide insights for efforts to improve the household management of childhood diarrhoea in Nicaragua.
Child; Diarrhoea; Household management; Nicaragua
Molecular Dynamics simulations of the pentamidine-S100B complex, where two molecules of pentamidine bind per monomer of S100B, were performed in an effort to determine what properties would be desirable in a pentamidine-derived compound as an inhibitor for S100B. These simulations predicted that increasing the linker length of the compound would allow a single molecule to span both pentamidine binding sites on the protein. The resulting compound, SBi4211 (also known as heptamidine), was synthesized and experiments to study its inhibition of S100B were performed. The 1.65 Å X-ray crystal structure was determined for Ca2+-S100B-heptamdine and gives high-resolution information about key contacts that facilitate the interaction between heptamidine and S100B. Additionally, NMR HSQC experiments with both compounds show that heptamidine interacts with the same region of S100B as pentamidine. Heptamidine is able to selectively kill melanoma cells with S100B over those without S100B, indicating that its binding to S100B has an inhibitory effect and that this compound may be useful in designing higher-affinity S100B inhibitors as a treatment for melanoma and other S100B-related cancers.
Molecular dynamics simulations of the pentamidine–S100B
complex, where two molecules of pentamidine bind per monomer of S100B,
were performed in an effort to determine what properties would be
desirable in a pentamidine-derived compound as an inhibitor for S100B.
These simulations predicted that increasing the linker length of the
compound would allow a single molecule to span both pentamidine binding
sites on the protein. The resulting compound, SBi4211 (also known
as heptamidine), was synthesized, and experiments to study its inhibition
of S100B were performed. The 1.65 Å X-ray crystal structure was
determined for Ca2+–S100B–heptamdine and
gives high-resolution information about key contacts that facilitate
the interaction between heptamidine and S100B. Additionally, NMR HSQC
experiments with both compounds show that heptamidine interacts with
the same region of S100B as pentamidine. Heptamidine is able to selectively
kill melanoma cells with S100B over those without S100B, indicating
that its binding to S100B has an inhibitory effect and that this compound
may be useful in designing higher affinity S100B inhibitors as a treatment
for melanoma and other S100B-related cancers.
structure-based discovery; pentamidine-related inhibitor; calcium-binding protein S100B
The goal of this study was to determine the degree to which the persistence of cryptococcosis, overall 1-year mortality, and 1-year mortality due to cryptococcosis were influenced by initial antifungal treatment regimen in a cohort of adults with cryptococcosis treated at a tertiary care medical center. Risk factors, underlying conditions, treatment, and mortality information were obtained for 204 adults with cryptococcosis from Duke University Medical Center (DUMC) from 1996 to 2009. Adjusted risk ratios (RR) for persistence and hazard ratios (HR) for mortality were estimated for each exposure. The all-cause mortality rate among patients with nonsevere disease (20%) was similar to that in the group with disease (26%). However, the rate of cryptococcosis-attributable mortality with nonsevere disease (5%) was much lower than with severe disease (20%). Flucytosine exposure was associated with a lower overall mortality rate (HR, 0.4; 95% confidence interval [CI], 0.2 to 0.9) and attributable mortality rate (HR, 0.5; 95% CI, 0.2 to 1.2). Receiving a nonrecommended antifungal regimen was associated with a higher relative risk of persistent infection at 4 weeks (RR, 1.9; 95% CI, 0.9 to 4.3), and the rate of attributable mortality among those not receiving the recommended dose of initial therapy was higher than that of those receiving recommended dosing (HR, 2.3; 95% CI, 1.0 to 5.0). Thus, the 2010 Infectious Diseases Society of America (IDSA) guidelines are supported by this retrospective review as a best-practice protocol for cryptococcal management. Future investigations should consider highlighting the distinction between all-cause mortality and attributable mortality so as not to overestimate the true effect of cryptococcosis on patient death.
S100A4, a member of the S100 family of Ca2+-binding proteins, modulates the motility of both non-transformed and cancer cells by regulating the localization and stability of cellular protrusions. Biochemical studies have demonstrated that S100A4 binds to the C-terminal end of the myosin-IIA heavy chain coiled-coil and disassembles myosin-IIA filaments; however, the mechanism by which S100A4 mediates myosin-IIA depolymerization is not well understood.
We determined the X-ray crystal structure of the S100A4Δ8C/MIIA1908-1923 peptide complex, which showed an asymmetric binding mode for the myosin-IIA peptide across the S100A4 dimer interface. This asymmetric binding mode was confirmed in NMR studies using a spin-labeled myosin-IIA peptide. In addition, our NMR data indicate that S100A4Δ8C binds the MIIA1908-1923 peptide in an orientation very similar to that observed for wild-type S100A4. Studies of complex formation using a longer, dimeric myosin-IIA construct demonstrated that S100A4 binding dissociates the two myosin-IIA polypeptide chains to form a complex composed of one S100A4 dimer and a single myosin-IIA polypeptide chain. This interaction is mediated, in part, by the instability of the region of the myosin-IIA coiled-coil encompassing the S100A4 binding site.
The structure of the S100A4/MIIA1908-1923 peptide complex has revealed the overall architecture of this assembly and the detailed atomic interactions that mediate S100A4 binding to the myosin-IIA heavy chain. These structural studies support the idea that residues 1908–1923 of the myosin-IIA heavy chain represent a core sequence for the S100A4/myosin-IIA complex. In addition, biophysical studies suggest that structural fluctuations within the myosin-IIA coiled-coil may facilitate S100A4 docking onto a single myosin-IIA polypeptide chain.
X-ray crystallography; NMR; S100A4; Myosin-II; Cytoskeleton; Coiled-coil
Malignant melanoma continues to be an extremely fatal cancer due to a lack of viable treatment options for patients. The calcium-binding protein S100B has long been used as a clinical biomarker, aiding in malignant melanoma staging and patient prognosis. However, the discovery of p53 as a S100B target and the consequent impact on cell apoptosis redirected research efforts towards the development of inhibitors of this S100B–p53 interaction. Several approaches, including computer-aided drug design, fluorescence polarization competition assays, NMR, x-ray crystallography and cell-based screens have been performed to identify compounds that block the S100B–p53 association, reactivate p53 transcriptional activities and induce cancer cell death. Eight promising compounds, including pentamidine, are presented in this review and the potential for future modifications is discussed. Synthesis of compound derivatives will likely exhibit increased S100B affinity and mimic important S100B–target dynamic properties that will result in high specificity.
Mutations in the second EF-hand (D61N, D63N, D65N, E72A) of S100B were used to study its Ca2+-binding and dynamic properties in the absence and presence of abound target, TRTK-12. With D63NS100B as an exception (D63NKD = 50 ± 9 µM), Ca2+-binding to EF2-hand mutants were reduced by more than 8-fold in the absence of TRTK-12 (D61NKD = 412 ± 67 µM; D65NKD = 968 ± 171 µM; E72AKD = 471 ± 133 µM), when compared to wild-type protein (WTKD = 56 ± 9 µM). For the TRTK-12 complexes, the Ca2+-binding affinity to wild type (WT+TRTKKD = 12 ± 10 µM) and the EF2 mutants were increased by 5- to 19-fold versus in the absence of target (D61N+TRTKKD = 29 ± 1.2 µM; D63N+TRTKKD = 10 ± 2.2 µM; D65N+TRTKKD = 73 ± 4.4 µM; E72A+TRTKKD = 18 ± 3.7 µM). In addition, Rex, as measured using relaxation dispersion for side chain 15N resonances of Asn63 (D63NS100B) was reduced upon TRTK-12 binding when measured by nuclear magnetic resonance (NMR). Likewise, backbone motions on multiple time scales (ps-ms) throughout wild type, D61NS100B D63NS100B, and D65NS100B were lowered upon binding TRTK-12. However, the X-ray structures of Ca2+-bound (2.0 Å) and TRTK-bound (1.2 Å) D63NS100B showed no change in Ca2+ coordination, so these and analogous structural data for the wild-type protein could not be used to explain how target binding increased Ca2+-binding affinity in solution. Thus, a model for how S100B-TRTK12 complex formation increases Ca2+ binding is discussed, which considers changes in protein dynamics upon binding the target TRTK-12.
NMR; relaxation dispersion; 15N relaxation; calcium-binding proteins; EF-hand; X-ray crystallography; S100 proteins; S100B
Gram-negative bacterial bloodstream infection (BSI) is a serious condition with estimated 30% mortality. Clinical outcomes for patients with severe infections improve when antibiotics are appropriately chosen and given early. The objective of this study was to estimate the association of prior healthcare exposure on time to appropriate antibiotic therapy in patients with gram-negative BSI.
We performed a multicenter cohort study of adult, hospitalized patients with gram-negative BSI using time to event analysis in nine community hospitals from 2003-2006. Event time was defined as the first administration of an antibiotic with in
vitro activity against the infecting organism. Healthcare exposure status was categorized as community-acquired, healthcare-associated, or hospital-acquired. Time to appropriate therapy among groups of patients with differing healthcare exposure status was assessed using Kaplan-Meier analyses and multivariate Cox proportional hazards models.
The cohort included 578 patients with gram-negative BSI, including 320 (55%) healthcare-associated, 217 (38%) community-acquired, and 41 (7%) hospital-acquired infections. 529 (92%) patients received an appropriate antibiotic during their hospitalization. Time to appropriate therapy was significantly different among the groups of healthcare exposure status (log-rank p=0.02). Time to first antibiotic administration regardless of drug appropriateness was not different between groups (p=0.3). The unadjusted hazard ratios (HR) (95% confidence interval) were 0.80 (0.65-0.98) for healthcare-associated and 0.72 (0.63-0.82) for hospital-acquired, relative to patients with community-acquired BSI. In multivariable analysis, interaction was found between the main effect and baseline Charlson comorbidity index. When Charlson index was 3, adjusted HRs were 0.66 (0.48-0.92) for healthcare-associated and 0.57 (0.44-0.75) for hospital-acquired, relative to patients with community-acquired infections.
Patients with healthcare-associated or hospital-acquired BSI experienced delays in receipt of appropriate antibiotics for gram-negative BSI compared to patients with community-acquired BSI. This difference was not due to delayed initiation of antibiotic therapy, but due to the inappropriate choice of antibiotic.
Rotavirus vaccines are highly effective at preventing gastroenteritis in young children and are now universally recommended for infants in the US. We studied patterns of use of rotavirus vaccines among US infants with commercial insurance. We identified a large cohort of infants in the MarketScan Research Databases, 2006–2010. The analysis was restricted to infants residing in states without state-funded rotavirus vaccination programs. We computed summary statistics and used multivariable regression to assess the association between patient-, provider-, and ecologic-level variables of rotavirus vaccine receipt and series completion. Approximately 69% of 594,117 eligible infants received at least one dose of rotavirus vaccine from 2006–2010. Most infants received the rotavirus vaccines at the recommended ages, but more infants completed the series for monovalent rotavirus vaccine than pentavalent rotavirus vaccine or a mix of the vaccines (87% versus 79% versus 73%, P<0.001). In multivariable analyses, the strongest predictors of rotavirus vaccine series initiation and completion were receipt of the diphtheria, tetanus and acellular pertussis vaccine (Initiation: RR = 7.91, 95% CI = 7.69–8.13; Completion: RR = 1.26, 95% CI = 1.23–1.29), visiting a pediatrician versus family physician (Initiation: RR = 1.51, 95% CI = 1.49–1.52; Completion: RR = 1.13, 95% CI = 1.11–1.14), and living in a large metropolitan versus smaller metropolitan, urban, or rural area. We observed rapid diffusion of the rotavirus vaccine in routine practice; however, approximately one-fifth of infants did not receive at least one dose of vaccine as recently as 2010. Interventions to increase rotavirus vaccine coverage should consider targeting family physicians and encouraging completion of the vaccine series.
S100 proteins are markers for numerous cancers, and in many cases high S100 protein levels are a prognostic indicator for poor survival. One such case is S100B, which is overproduced in a very large percentage of malignant melanoma cases. Elevated S100B protein was more recently validated to have causative effects towards cancer progression via down-regulating the tumor suppressor protein, p53. Towards eliminating this problem in melanoma, targeting S100B with small molecule inhibitors was initiated. This work relies on numerous chemical biology technologies including structural biology, computer-aided drug design, compound screening, and medicinal chemistry approaches. Another important component of drug development is the ability to test compounds and various molecular scaffolds for their efficacy in vivo. This chapter briefly describes the development of S100B inhibitors, termed SBiXs, for melanoma therapy with a focus on the inclusion of in vivo screening at an early stage in the drug discovery process.
In vivo screening; Preclinical testing; Intratumoral delivery; Systemic delivery; Pharmacokinetics; Pharmacodynamics; Maximum tolerated dose; Therapeutic window; Genetically modified mouse models; S100 proteins; EF-hand
Membrane-associated serine protease matriptase has been implicated in human diseases, and might be a drug target. In the present study, a novel class of matriptase inhibitors targeting zymogen activation is developed by a combination of the screening of compound library using a cell-based matriptase activation assay and a computer-aided search of commercially available analogs of a selected compound. Four structurally related compounds are identified that can inhibit matriptase activation with IC50 at low μM in both intact-cell and cell-free systems, suggesting that these inhibitors target the matriptase autoactivation machinery rather than the intracellular signaling pathways. These activation inhibitors can also inhibit prostasin activation, a downstream event that occurs in lockstep with matriptase activation. In contrast, the matriptase catalytic inhibitor CVS-3983 at a concentration 300-fold higher than its Ki fails to inhibit activation of either protease. Our results suggest that inhibiting matriptase activation is an efficient way to control matriptase function.
Nicaragua was the first developing nation to implement universal infant rotavirus immunization with the pentavalent rotavirus vaccine (RV5). Initial studies of vaccine effectiveness in Nicaragua and other developing nations have focused on the prevention of hospitalizations and severe rotavirus diarrhea. However, rotavirus diarrhea is more commonly treated in the primary care setting, with only 1–3% of rotavirus cases receiving hospital care. We measured the prevalence of rotavirus infection in primary care clinics in León, Nicaragua, after introduction of the immunization program. In the post-vaccine period, 3.5% (95% confidence interval = 1.9–5.8) of children seeking care for diarrhea tested positive for rotavirus. A high diversity of rotavirus genotypes was encountered among the few positive samples. In conclusion, rotavirus was an uncommon cause of childhood diarrhea in this primary care setting after implementation of a rotavirus immunization program.
We hypothesized that cofilin activation by members of the slingshot (SSH) phosphatase family is a key mechanism regulating VSMC migration and neoinitima formation following vascular injury.
Methods and Results
Scratch wound and modified Boyden chamber assays were used to assess VSMC migration following downregulation of the expression of cofilin and each slingshot phosphatase isoforms (SSH1,-2,-3) by siRNA, respectively. Cofilin siRNA greatly attenuated the ability of VSMC migration into the “wound” and PDGF-induced migration was virtually eliminated versus a 3.5-fold increase in non-treated VSMCs, establishing a critical role for cofilin in VSMC migration. Cofilin activation (dephosphorylation) was increased in PDGF-stimulated VSMCs. Thus, we assessed the role of the SSH family of phosphatases on cofilin activation and VSMC migration. Treatment with either SSH1 or SSH2 siRNA attenuated cofilin activation, while SSH3 siRNA had no effect. Only SSH1 siRNA significantly reduced wound healing and PDGF-induced VSMC migration. Both SSH1 (4.7 fold) and cofilin (3.9 fold) expression were increased in balloon injured versus non-injured carotid arteries and expression was prevalent in the neointima.
These studies demonstrate that the regulation of VSMC migration by cofilin is SSH1 dependent, and that this mechanism potentially contributes to neointima formation following vascular injury in vivo.
PDGF; VSMC migration; vascular injury; slingshot phosphatase; neointima